Research Article

Xun Gao*#, Kai Xu#, Miaomiao Chi, Jiaojiao Li, Lingzhe Suo, Lin Zhu, Kexin Chen, and Jingqing Mu

Determination of four parabens in cosmetics byhigh-performance liquid chromatography withmagnetic solid-phase and ionic dispersiveliquid–liquid extraction

https://doi.org/10.1515/revac-2021-0133received November 22, 2020; accepted April 07, 2021

Abstract: To determine the trace amount of four benzoicacid esters in cosmetics, ionic dispersive liquid–liquidmicroextraction (DLLME) and magnetic solid-phase extrac-tion were combined and optimized. After solvent optimiza-tion, 1-octyl-3-methylimidazoliumhexafluorophosphatewasselected as the extraction solvent to form hydrophobic dro-plets in the process of ionic DLLME, followed by removal ofions from the sample solution containing Fe3O4@GO nano-

materials. The magnetic nano-materials were characterizedusing Fourier transform infrared spectroscopy, scanningelectron microscopy, transmission electron microscopy,and vibrating sample magnetometer. Some parametersaffecting the efficiency of extraction were optimized usingBox-Behnken design. Under optimized conditions, thelimit of detection for all the preservatives was less than0.135mg/L and the accuracy ranged from 88.5% to 101%.This technology could determine the trace amount of pre-servatives in cosmetics with comparatively higher accu-racy and sensitivity.

Keywords: preservatives, ionic liquids, dispersive liquid–liquid microextraction, magnetic solid-phase extraction

Abbreviations

[C8MIM][PF6] 1-octyl-3-methylimidazoliumhexafluorophosphate

ANOVA analysis of varianceBBD Box-Behnken designBP butyl-parabenC.V. coefficient of variationCE capillary electrophoresisCPE cloud point extractionDLLME dispersive liquid–liquid microextractionEP ethyl 4-hydroxybenzoateFe3O4@GO magnetic graphene oxideFLC fast liquid chromatographyFT-IR Fourier transform infrared spectroscopyGO graphene oxideHPLC high-performance liquid

chromatographyILs ionic liquidsLOD limit of detectionLOQ limit of quantificationMAE microwave-assisted extraction

* Corresponding author: Xun Gao, School of Pharmacy, Jiangsu KeyLaboratory of Marine Pharmaceutical Compound Screening, JiangsuOcean University, 59 Cangwu Road, Haizhou District, Lianyungang,222001, China; Co-Innovation Center of Jiangsu Marine Bio-industryTechnology, Jiangsu Ocean University, 59 Cangwu Road, Lianyungang,222001, China, e-mail: gaoxun_2017@163.com,tel: +8613555824733Kai Xu: School of Pharmacy, Jiangsu Key Laboratory of MarinePharmaceutical Compound Screening, Jiangsu Ocean University,59 Cangwu Road, Haizhou District, Lianyungang, 222001, China;Co-Innovation Center of Jiangsu Marine Bio-industry Technology,Jiangsu Ocean University, 59 Cangwu Road, Lianyungang, 222001,China; Jiangsu Institute of Marine Resources Development,59 Cangwu Road, Lianyungang, 222005, ChinaMiaomiao Chi, Lin Zhu, Kexin Chen, Jingqing Mu: School ofPharmacy, Jiangsu Key Laboratory of Marine PharmaceuticalCompound Screening, Jiangsu Ocean University, 59 Cangwu Road,Haizhou District, Lianyungang, 222001, ChinaJiaojiao Li: School of Pharmacy, Jiangsu Key Laboratory of MarinePharmaceutical Compound Screening, Jiangsu Ocean University,59 Cangwu Road, Haizhou District, Lianyungang, 222001, China;Co-Innovation Center of Jiangsu Marine Bio-industry Technology,Jiangsu Ocean University, 59 Cangwu Road, Lianyungang, 222001,ChinaLingzhe Suo: Hangzhou Sino-American Huadong Medicines Co.,Ltd. Hangzhou, 310011, China

# These authors contributed equally to this work.

Reviews in Analytical Chemistry 2021; 40: 161–172

Open Access. © 2021 Xun Gao et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 InternationalLicense.

MP methyl 4-hydroxybenzoateMSPE magnetic solid-phase extractionPP propyl p-hydroxybenzoateRSDs relative standard deviationSEM scanning electron microscopeSPE solid-phase extractionTEM transmission electron microscopyUAE ultrasonic-assisted extractionUHPLC ultra-high-performance liquid

chromatographyVSM vibrating sample magnetometer

1 Introduction

Currently, most of the active ingredients in cosmetics arenutrients, which may degrade easily because of microbialcontamination, failing product specification [1]. Becauseof their comparatively wider antibacterial spectrum, strongantibacterial activity, and good solubility in water, andbecause they are colorless, odorless, and cheaper, benzoatepreservatives are widely used in the production of cosmetics[2–5]. According to recent medical research results, exces-sive paraben may cause irritation to the human skin andrespiratory and endocrine systems [6–9]. In addition, expo-sure to paraben in some cosmetic products has been provedto be associated with birth size [10]. The safety issue ofparaben in cosmetics has attracted increasing attention,and in many countries, the accepted limits of preservativesare strictly controlled. Taking p-hydroxybenzoic acid as anexample, the authorized maximum content of single estersand ester mixtures should not exceed 0.4% (v/v) and 0.8%(v/v), respectively [11].

As the formulations of cosmetic products are rela-tively complicated and diverse, the reported methodsshowed comparatively low sensitivity, accuracy, and effi-ciency [12]. Thus, sample preparation, including precon-centration and purification, are the key steps to improveaccuracy and sensitivity when using high-performanceliquid chromatography (HPLC) and ultra-high-performanceliquid chromatography (UHPLC) based on the ultraviolet-visible detector and diode array detector [13–16].

Different extraction technologies for benzoate preser-vative have been reported, such as vortex extraction,supercritical fluid extraction, matrix solid-phase disper-sion, and quick, easy, cheap, effective, rugged, and safe.To reduce energy cost and organic solvent usage withoutcompromising extraction efficiency, dispersive liquid–liquid microextraction (DLLME) was optimized to con-centrate the target analytes in extracting solvent [17].Furthermore, ionic liquids (ILs), known as green sol-vents, were used as extracting solvents in ILs-DLLME

with their unique properties, such as high chemicaland thermal stability, low flammability, and excellentsolvation ability for a wide range of compounds [18–20].Moreover, to avoid time-consuming centrifugation in ILs-DLLME, the magnetic nano-materials were applied toabsorb the target analytes by hydrophobic interactionwhich was known as magnetic solid-phase extraction(MSPE). Magnetic graphene oxide (Fe3O4@GO) nano-mate-rials, with high extraction ability and its magnetic proper-ties, could improve efficiency in purification [21–23,16].

In this paper, IL-DLLME-MSPE, which was a methodrarely reported in the literature, was developed for theextraction of four types of benzoic acid esters from cos-metics. IL-DLLME remarkably cut the energy and cost byreducing the usage of organic solvents and improved theease of operation, and MSPE greatly improved the effi-ciency of separation and extraction, which solved theproblem of long-time centrifugation, freezing, or manualcollection of large amounts of organic solvents in DLLME.The optimized method was proved to be easy operating,cost-effective, and eco-friendly with acceptable accuracyand precision, which can be applied in the routine assayfor preservatives in cosmetics [24–26].

2 Experimental

2.1 Instruments

HPLC analysis was performed using Agilent 1200 highperformance liquid chromatograph (California,USA) equippedwith a Diamonsil C18 column (4.6mm × 150mm, 5 μm)(Beijing, China). The column temperature was kept at35°C. The injection volume was set at 10 μL. The analyteswere separated using isocratic elution with a 70:30 (v/v)mixed solution of methanol and water at a flow rate of0.8mL/min. The detection wavelength was set at254 nm. FT-IR spectra were recorded on a Bruker IFS-66Fourier transform infrared spectroscopy (Karlsruhe,Germany). Magnetization measurements were performedon a Quantum Design Vibrating Sample Magnetometer(VSM, San Diego, USA). The structure and morphologyof the prepared nano-materials were characterized by aHelio Nanolab 600i Scanning Electron Microscope (SEM,Hillsboro, USA). The transmission electron microscopy(TEM) images of the prepared nano-materials were obtainedusing a JEM-2100F electron microscopy (Tokyo, Japan).

2.2 Materials

Deionized water was obtained from Wahaha GroupCo., Ltd. (Hangzhou, China). HCl, NaOH, and absolute

162 Xun Gao et al.

alcohol (purity, 95%)were supplied by Hengxing ChemicalReagent Factory (Tianjin, China). Chromatographic puremethanol, acetonitrile, and acetone were purchased fromShandong Yuwang Industrial Co., Ltd. (Yucheng, China).FeCl3·6H2O (purity, 99%) and FeCl2·4H2O (purity, 99%)were provided by Ailan Chemical Technology Co., Ltd.(Shanghai, China). Graphene oxide (GO) was supplied byNanjing Ji Cang Nano Technology Co., Ltd. (Nanjing,China). Methyl 4-hydroxybenzoate (MP), ethyl 4-hydro-xybenzoate (EP), propyl p-hydroxybenzoate (PP), butyl-paraben (BP) standard, and 1-octyl-3-methylimidazoliumhexafluorophosphate ([C8MIM][PF6]) were purchasedfrom Shanghai Aladdin Chemistry Co., Ltd. (Shanghai,China). The standard solutions of MP, EP, PP, and BPwith a concentration of 100 μg/mL were prepared ina volumetric flask and stored away from light at 4°C.The standard solutions were diluted with methanol andprepared every day.

2.3 Preparation of Fe3O4@GO

nano-materials

According to the chemical co-precipitation method,Fe3O4@GO nano-materials were prepared [27]. Briefly,0.5 g of GO powder and 100mL of distilled water weretaken in a 250mL three-neck round bottom flask, fol-lowed by mechanical stirring until the water bath tem-perature increased evenly from 20°C to 70°C in 3 min.About 2.16 g of FeCl3·6H2O and 0.80 g of FeCl2·4H2Owere weighed and dissolved in 40mL of distilled waterwith vigorous stirring at 70°C for 5 min. Then the pH ofthis suspension was adjusted to 12.0 with an ammoniasolution (28%, g/mL) to conjugate the Fe3O4 nano-mate-rials onto GO sheets. After mechanically stirring at 70°Cfor 60min, the black magnetic material was separatedfrom the suspension using a magnet and washed thricewith distilled water and ethanol. To avoid completegrowth and oxidation of the nano-material crystals, theabove reaction needs to be carried out in a nitrogen envir-onment. Fe3O4@GO nano-materials were achieved afterdrying under vacuum at 60°C for 6 h and grounded usinga mortar for further use.

2.4 Sample preparation

We have purchased five different brands of cosmetics froma local shopping mall (Shenyang, Liaoning), includinglotion and facial cleanser. Weighed 5 g of lotion and 5 gof facial cleanser, and measured 1 mL of toning lotion

and 1 mL of cleansing water to 100 mL centrifugal tubeseparately. This was mixed with 50mL of distilled waterand the matrix dispersed by vortexing for 2min. Aftersonication for 10min, the suspension was filtered througha 0.45 μm filter membrane and stored at 4°C for furtheruse.

2.5 Pretreatment of IL-DLLME and MSPE

About 4.0 g of NaCl was weighed and dissolved comple-tely in the above samples, and the pH was adjusted to 3.2with 0.1mol/L HCl. Then, 30 μL of [C8MIM][PF6] and aceto-nitrile were added to the suspension before shakingfor 2 min. After that, 32.7 mg of Fe3O4@GO was addedto the dispersion solution, and sonicated for 6 min.After separation by a magnet, magnetic nano-materialswere resuspended in 2 mL of absolute ethanol and soni-cated for 5 min. Finally, Fe3O4@GO were magneticallyseparated from the suspension again, and the super-natant was collected and filtered with a 0.22 μm filter mem-brane before injecting into HPLC for further analysis.

3 Results and discussion

3.1 Characterization of Fe3O4@GOnanoparticles

The dimension and surface morphologies of the nano-materials were characterized by SEM, TEM, FT-IR, and VSM.

The surface morphologies of GO and Fe3O4@GO fromSEM are shown in Figure 1a and b. Figure 1a show atypically smooth and disorderly folded sheet-like struc-ture of GO. In comparison, the SEM image of Fe3O4@GOin Figure 1b show a rougher surface because a largenumber of Fe3O4 nano-materials have been attached tothe surface of GO. The structural characteristics of GOand Fe3O4@GO observed by TEM are shown in Figure1c, and the GO was sheet-like in shape with high trans-parency and wrinkled edges. Fe3O4 nano-materials werelocated at the cross-section of Fe3O4@GO nano-materials(Figure 1d) and observed as dark spots that distributedhomogeneously on the smooth surface of the GO sheet.

Figure 2 shows the FT-IR spectra of GO and Fe3O4@GO.The most distinct peak at 3436.6 cm−1 was attributed to theO–H stretching vibration. The characteristic peaks of GOthat appeared at 1720.8, 1629.6, 1401.1, and 1052.1 cm−1 werecorresponded to the carbonyl (C]O) stretching, sp2 carbonskeletal network, C–OH group stretching, and C–O–C in theepoxide group stretching vibration, respectively. According to

Application of various techniques for sample preparation of cosmetics 163

the spectra of MGO, the new presence of absorption peak at1396.4 cm−1 indicated an additional vibration band in sam-ples, confirming the formation of the chemical bond betweenthe carboxyl group and Fe. In addition, the peak at 584.5 cm−1

was attributed to the Fe–O–Fe bond vibration. Consideringthe above evidence, we could conclude that the covalentbonds betweenmagnetic nanoparticles and GOwere success-fully synthesized.

The magnetic property of Fe3O4@GO at 20°C wasinvestigated using VSM, and the hysteresis loop diagramis shown in Figure 3. The magnetization hysteresis loopsof Fe3O4@GO were S-like curves, indicating a superpara-magnetic capability of the synthesized nano-materials. Thesaturate magnetization value of Fe3O4@GO was 47.15 emu/gand enough to be separated with conventional magnets thatwere sufficient for magnetic separation with conventionalmagnets. Therefore, Fe3O4@GO was able to be separatedrapidly from the aqueous matrix.

3.2 Optimization of IL-DLLME

To obtain optimum extraction efficiency of preservativesin cosmetics, a series of experiments were designed.

Some parameters that may affect the extraction effi-ciency, such as vortexing time, ionic strength, the volumeof extraction solvent, and disperser solvent, were opti-mized. The types of extraction solvent and disperser sol-vent have been selected according to the previous studywith modifications [18].

Figure 1: (a) SEM image of GO; (b) SEM image of Fe3O4@GO; (c) TEM image of GO; and (d) TEM image of Fe3O4@GO.

Figure 2: FT-IR spectrum of GO and Fe3O4@GO.

164 Xun Gao et al.

3.2.1 Volume optimization of extraction solvent

The volume of ILs also played a critical role in improvingthe extraction efficiency in IL-DLLME. The volume of theextraction solvent varied from 10 to 50 μL. As shown inFigure 4a, the recoveries decreased slightly when thevolume was more than 30 μL. This may be because theexcessive volume of [C8MIM][PF6] was easier to adhere tothe side-walls of tubes and could not effectively extract thetarget analytes in the form of tiny droplets. Therefore, thevolume of 30 μL of [C8MIM][PF6] was selected.

3.2.2 Volume optimization of disperser solvent

The influence of the acetonitrile volume on the extractionefficiency of the four common preservatives in cosmeticswas also investigated in the range of 10–50 μL. When theextraction solvent is less than 30 μL, the extraction

Figure 3: Magnetization curves of GO and Fe3O4@GO.

Figure 4: Optimization of extraction parameters for IL-DLLME including (a) volume of ionic liquid, (b) volume of disperser solvent, (c) time ofvortex, and (d) amount of NaCl. In each optimization, the optimal amount of the previous step was used. Other conditions were consideredunchanged and the same as the previous step.

Application of various techniques for sample preparation of cosmetics 165

solvent unable to be evenly dispersed in the water phasein the form of droplets, however, when the volume ofdisperser solvent is higher than 30 μL, the solubilityof the analyte in the aqueous phase and decrease itsconcentration in the extraction solvent, both of whichresulted in poor recovery. Therefore, given the considera-tions of cost and recovery in Figure 4b, we selected thevolume of 30 μL for acetonitrile in further study.

3.2.3 Optimization of vortexing time

The vortexing time is an important factor in the efficiencyof dispersing cosmetic samples, and the time rangedfrom 0 to 5 min was investigated. The results are shownin Figure 4c; when the vortex time was 2 min, the recov-eries of the four common preservatives were more than90%. However, with the increase in time, the recoveriesdecreased slightly or basically unchanged. Therefore,vortexing for 2 min was used to the next study.

3.2.4 Optimization of ionic strength

In IL-DLLME, the ionic strength can reduce the solubilityof target analytes in the aqueous phase and increase the

Table 1: Analysis of variance (ANOVA) test for the second-orderregression model

Source Sum ofsquares

Degreeoffreedom

Meansquare

F-value p-value(Prob > F)

Model 6408.17 9 712.02 17.26 0.0005*significant

A-pH 3969.40 1 3969.40 96.25 <0.0001*B-Sorbent 335.41 1 335.41 8.13 0.0246C-time 420.50 1 420.50 10.20 0.0152AB 61.62 1 61.62 1.49 0.2611AC 97.02 1 97.02 2.35 0.1689BC 97.02 1 97.02 2.35 0.1689A2 489.98 1 489.98 11.88 0.0107B2 677.78 1 677.78 16.43 0.0048C2 126.79 1 126.79 3.07 0.1230Residual 288.68 7 41.42Lack of fit 198.90 3 66.30 2.95 0.1613 not

significantPure error 89.78 4 22.45Cor total 6696.86 16

R2 =95.69%

Adj R2 =90.15%

C.V.% =8.89

*Significant at 0.01 level.

Figure 5: Response surface plots for effect of the MSPE extraction for (a) extraction time, (b) amount of sorbent, and (c) pH.

166 Xun Gao et al.

Figure 6: Optimization of elution parameters for MSPE including (a) type of desorption solvent, (b) volume of desorption, and (c) time ofdesorption.

Figure 7: Chromatograms of the mixed reference solution.

Application of various techniques for sample preparation of cosmetics 167

partition coefficient of the [C8MIM][PF6] phase andfurther affect the recovery of extraction. The influenceof ionic strength in this experiment was assessed by theaddition of NaCl in the range of 0–6.0 g in initial aqueoussample. Figure 4d shows that the recoveries reached themaximum value when 4.0 g of NaCl was used. However,excessive salt could also increase the solubility of ILsin the aqueous phase, resulting in a decrease in therecoveries.

3.3 Optimization of MSPE conditions

To understand the influence of experimental variables onthe MSPE process, the interaction between the main para-meters that affect the extraction efficiency of the analytemust be fully considered, and each parameter must beoptimized. On the basis of preliminary experiments, theinfluence of six factors was studied from two levels ofextraction process and elution process. The main para-meters, such as pH of the sample solution, extractiontime, and amount of sorbent, were evaluated using theBox-Behnken design (BBD) with a small number of trials,which is a widely used method to fit a second-orderresponse surface. The other three parameters, such astype and volume of desorption solvent, and desorptiontime, were optimized by one-factor-at-a-time procedure.

The Design-Expert 11.0 statistical software was usedfor the experimental design and data analysis. Basedon the results acquired from the analysis of variance(ANOVA) test (presented in Table 1), the significance ofmodel is mainly calculated by R2 value. As presented inTable 1, the coefficients were very high about 0.9569,which indicated that 95.69% of total variance wereexplained by quadratic model and only 4.31% of totalvariables were not explained by the model. The adjustedR2 value was 0.9015. The R2 and adjusted R2 values wereclosed to 1.0000, which displayed a good correlationbetween experimental and theoretical results. The modelhad F values of 17.26 with lower p-values (0.0005), whichalso indicated the impacts of this model. If the lack offit value in a model was nonsignificant, it suggestedthat the quadratic model was valid. Here, the lack of fitvalues were 0.1613, which specified that our model wassignificant. Another one was the coefficient of variation(C.V.%); if this value was less than 10%, then the modelwas highly reliable and reproducible for the experimentalstudies. The C.V.% value for clenbuterol was 8.89%, lessthan 10%. Therefore, the model was reliable for experi-mental studies for the determination of four preservativesin cosmetics.

The pH of the sample solution has a negative effecton the recovery in MSPE process because it can determine

Table 2: The analytical performance data for HPLC system

Analytes Retention time (min) Calibration equation Linear range (mg/L) R2 LOQ (mg/L) LOD (mg/L)

MP 3.290 Y = 72.29X + 63.97 0.5–100 0.9997 0.216 0.064EP 3.995 Y = 67.73X + 52.63 0.5–100 0.9995 0.228 0.068PP 5.233 Y = 64.05X + 47.70 0.5–100 0.9989 0.231 0.069BP 7.296 Y = 58.55X + 41.88 0.5–100 0.9980 0.451 0.135

Table 3: Intra-day and inter-day recoveries and relative repeat-ability standard deviation of analytes in cosmetic samples

Analytes Spiked(mg/L)

Inter-day (n = 3) Intra-day (n = 6)

Relativerecovery(%)

RSD (%) Relativerecovery(%)

RSD (%)

MP 1 88.3 2.1 85.6 4.510 87.9 2.8 86.6 3.950 84.1 1.7 84.2 3.2

EP 1 93.2 4.3 90.2 4.210 92.3 2.4 91.8 3.850 89.7 2.9 89.3 1.7

PP 1 99.2 3.7 98.4 5.610 98.6 2.9 97.7 3.150 97.3 1.5 97.8 1.2

BP 1 96.7 2.1 96.8 4.710 95.3 2.6 96.1 2.250 93.7 1.8 94.6 0.9

Table 4: Concentrations of four parabens in real samples

Sample Content of the analytes (mg/kg)

MP EP PP BP

Astringent 1 138.2 74.53 48.13 103.3Astringent 2 ND ND ND NDCleansing water 1 4,290 ND ND NDCleansing water 2 6.422 ND ND NDLotion 272.7 ND 0.9510 NDFacial cleanser ND ND ND 31.04

ND: not detected.

168 Xun Gao et al.

the ionization of the target analytes and further affecttheir recoveries. It is seen from Figure 5 that as the pHincreases, the recoveries of four benzoates become moreunfavorable. When the pH value was higher than the pKa

of benzoate, the target analytes existed in the ionizedform could hardly be extracted using organic solvents.The extraction efficiency decreased with the hydrolysisof benzoate at a pH of 3.2, and the results in Figure 5show that the amounts of Fe3O4@GO was positive to effi-ciencies of MSPE in the range of 10–32.7 mg. To ensuresufficient contact between the adsorbent and the targetanalytes, the relative recoveries that are shown in Figure 5increased with the increase in the extraction time from1 to 5 min and then remained plateau after 5 min.

To elute the adsorbed analytes off the adsorbent com-pletely, eluting conditions, such as the type and volumeof solvent, as well as the time for desorption, were

evaluated to improve the recovery. Three organic solvents,methanol, acetonitrile, and ethanol, were investigated. Itcan be seen from Figure 6 that ethanol generated thehighest recovery of analytes. The volume of the desorptionsolvent was also investigated in the range of 3.0–9.0mL.As shown in Figure 6, the results indicated that 2.0mL ofethanol was sufficient to elute all the target analytes withan acceptable recovery. In addition, the desorption time inthe range of 1–11min was compared. As shown in Figure 6,benzoate could be efficiently eluted in 5min.

3.4 Validation of the method

Under the optimal experimental conditions, the linearity,precision, accuracy, sensitivity, and specificity of themethodwere validated.

Mixed reference solution, blank solution, and testsolution were injected into HPLC for analysis. The results(Figure 7) show that no interference was observed.

Within the range of 0.5–100 μg/mL for the standardsolution, 0.5, 1, 5, 10, 20, 50, and 100 μg/mL of the stan-dard solution in the sequence was added to 0.5 g placebo(cosmetic milk) and introduced into HPLC for analysisafter sample preparation. The criteria for the limit ofdetection (LOD) were set at the signal-to-noise ratio of3, and the limit of quantification (LOQ) was set at 10.

The linearity of all analytes showed a good correla-tion in the specified range of 0.5–100mg/L and the R2

ranged from 0.9980 to 0.9997 in Table 2. Under the opti-mized condition, the LOD of the four target analytesranged from 0.064 to 0.135 μg/mL. The sensitivity ofthis method meets the analysis and detection require-ments of four preservatives in cosmetics [11].

Figure 8: Chromatogram of the real sample.

Figure 9: Comparison of pretreatment methods between DLLME-MSPE, DLLME, and MSPE.

Application of various techniques for sample preparation of cosmetics 169

Accuracy and precision were conducted by spikingthe samples with mixed standards at three different levels(n = 3 at each level), and six replicates of each concentra-tion were made in parallel for 3 days. The mixed referencesolution was added to a 0.5 g blank matrix to make finalconcentrations of 1, 10, and 50 μg/mL. The recoveryand relative standard deviations (RSDs) are presentedin Table 3, the intra-day RSDs for all the analytes wereno more than 4.3%, and the inter-day RSDs were no morethan 5.6%. The above results showed that the accuracyand precision of this method are good.

3.5 Assay of products

To verify the applicability of the optimized and validatedmethod, six different types of cosmetic products wereanalyzed, including paste, emulsion, and liquid formula.The results are presented in Table 4, and the chromato-gram of the real sample is shown in Figure 8. The “SafetyTechnical Specifications for Cosmetics” (2015 edition) sti-pulates that the total amount of parabens in cosmeticsshall not exceed 0.5%, and the amount of monomersadded shall not exceed 0.4% [28]. After analysis, mostof the four benzoate compounds in the cosmetic samplesdid not exceed the specified limit, and only one sample ofthe makeup remover was detected to be out of the speci-fication MP.

3.6 Comparison of pretreatment methods

The extraction recoveries of DLLME-MSPE were com-pared with DLLME or MSPE alone, and the results areshown in Figure 9. It was found that Fe3O4@GO nano-composites adsorbed and separated ILs in the samplesolution, greatly improving the separation and extractionefficiency, which solved the problem of long-time con-sumption by centrifugation ormanual collection. The newlydeveloped method possessed a wide linearity range, highaccuracy, and short sample preparation time, which pro-vides a powerful means for monitoring the trace amount ofpreservative compounds in cosmetics.

3.7 Comparison to reported methods

The comparison between the previously reported methodand our proposed method for analyzing MP, EP, PP, andTa

ble5:

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170 Xun Gao et al.

BP in different products is listed in Table 5. Identificationof the four parabens in cosmetics, human secretions, andenvironmental and food samples were reported but thesample preparation using IL-DLLME-MSPE has rarelybeen reported for the analysis of preservatives in cos-metics. Alshana et al. applied capillary electrophoresis(CE) to determine the four parabens in breast milk andfood samples, with an LOD of 0.1–0.2 μg/mL and recoveryof 86.7–103.3% [29]. For the reported ultrasonic-assistedextraction (UAE) combining fast liquid chromatography(FLC)method, the LOD of the four parabens in cosmetics,cleaning agents, and pharmaceutical products ranged in0.06–4.38 μg/mL [30]. Currently, for the analysis of pre-servatives, there is no universal sample preparation tech-nique and analytical method.

4 Conclusion

In this experiment, two-step extraction techniques ofIL-DLLME and MSPE were used, combined with HPLCmethod to carry out selective extraction enrichment andtrace determination of four benzoate preservatives in dif-ferent types of cosmetic samples. The optimized methodwas efficient, accurate, and environment-friendly. It canbe applied to analyze preservatives in cosmetics sampleswithout matrix interference. Compared with the tradi-tional DLLME method, [C8MIM][PF6] of ILs is a relativelygreen extractant that offered higher sensitivity and line-arity range. This method possessed accepted accuracy,linearity, precision, and detection limit for the determi-nation of benzoic esters in real samples. This newlydeveloped two-step extraction technology may have abroad application prospect in the analysis of trace pre-servatives in cosmetics.

Funding information: This work was supported by theNational Natural Science Foundation of China (No. 81503029)and Young and Middle-aged Backbone Personnel TrainingProgram of Shenyang Pharmaceutical University (ZQN2016011).

Author contributions: Kai Xu: writing– review and editing,conceptualization, funding acquisition, methodology; XunGao: conceptualization, formal analysis, funding acquisi-tion, resources, supervision; Miaomiao Chi: data curation,writing – original draft, project administration; Jiaojiao Li:supervision, software; Lingzhe Suo: data curation, valida-tion; Lin Zhu: software, validation; Kexin Chen: visualiza-tion; Jingqing Mu: investigation.

Conflict of interest: The authors state no conflict ofinterest.

Data availability: All data generated or analysed duringthis study are included in this published article.

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